Auxotrophic Yeast Strains in Fundamental and Applied Research

Pronk, Jack T.

doi:10.1128/aem.68.5.2095-2100.2002

Cited by 267 publications

(256 citation statements)

References 51 publications

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“…Shake-flask cultivation for the determination of intracellular accumulation of total carotenoids were performed as follows: Cells were inoculated in 500 ml flasks containing 100 ml defined synthetic medium (5 g/l (NH 4 ) 2 SO 4 , 3 g/l KH 2 PO 4 , 0.5 g/l MgSO 4 and trace elements) supplemented with 20 g/l glucose, vitamins and auxotrophic requirements histidine, leucine, methionine and uracil, as previously described (Pronk, 2002) with an initial pH of 6.0. The flasks were incubated at 30 • C on an orbital shaker set at 200 rpm.…”

Section: Strain Cultivationsmentioning

confidence: 99%

Heterologous carotenoid production in Saccharomyces cerevisiae induces the pleiotropic drug resistance stress response

Verwaal

Jiang

Wang

et al. 2010

Yeast

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To obtain insight into the genome-wide transcriptional response of heterologous carotenoid production in Saccharomyces cerevisiae, the transcriptome of two different S. cerevisiae strains overexpressing carotenogenic genes from the yeast Xanthophyllomyces dendrorhous grown in carbon-limited chemostat cultures was analysed. The strains exhibited different absolute carotenoid levels as well as different intermediate profiles. These discrepancies were further sustained by the difference of the transcriptional response exhibited by the two strains. Transcriptome analysis of the strain producing high carotenoid levels resulted in specific induction of genes involved in pleiotropic drug resistance (PDR). These genes encode ABC-type and major facilitator transporters which are reported to be involved in secretion of toxic compounds out of cells. β-Carotene was found to be secreted when sunflower oil was added to the medium of S. cerevisiae cells producing high levels of carotenoids, which was not observed when added to X. dendrorhous cells. Deletion of pdr10, one of the induced ABC transporters, decreased the transformation efficiency of a plasmid containing carotenogenic genes. The few transformants that were obtained had decreased growth rates and lower carotenoid production levels compared to a pdr5 deletion and a reference strain transformed with the same genes. Our results suggest that production of high amounts of carotenoids in S. cerevisiae leads to membrane stress, in which Pdr10 might play an important role, and a cellular response to secrete carotenoids out of the cell.

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Section: Strain Cultivationsmentioning

confidence: 99%

Heterologous carotenoid production in Saccharomyces cerevisiae induces the pleiotropic drug resistance stress response

Verwaal

Jiang

Wang

et al. 2010

Yeast

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“…Because previous studies (21, 87) had shown that starvation for auxotrophic requirements accelerates cell aging, these were provided in fivefold excess to planktonic cells. As the medium feeding ICRs was regularly replenished, auxotrophic requirements were provided there at recommended concentrations (88). Details of routine strain storage and maintenance are described in SI Materials and Methods.…”

Section: Methodsmentioning

confidence: 99%

Uncoupling reproduction from metabolism extends chronological lifespan in yeast

Nagarajan

Kruckeberg

Schmidt

et al. 2014

Proc. Natl. Acad. Sci. U.S.A.

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Studies of replicative and chronological lifespan in Saccharomyces cerevisiae have advanced understanding of longevity in all eukaryotes. Chronological lifespan in this species is defined as the agedependent viability of nondividing cells. To date this parameter has only been estimated under calorie restriction, mimicked by starvation. Because postmitotic cells in higher eukaryotes often do not starve, we developed a model yeast system to study cells as they age in the absence of calorie restriction. Yeast cells were encapsulated in a matrix consisting of calcium alginate to form ∼3 mm beads that were packed into bioreactors and fed ad libitum. Under these conditions cells ceased to divide, became heat shock and zymolyase resistant, yet retained high fermentative capacity. Over the course of 17 d, immobilized yeast cells maintained >95% viability, whereas the viability of starving, freely suspended (planktonic) cells decreased to <10%. Immobilized cells exhibited a stable pattern of gene expression that differed markedly from growing or starving planktonic cells, highly expressing genes in glycolysis, cell wall remodeling, and stress resistance, but decreasing transcription of genes in the tricarboxylic acid cycle, and genes that regulate the cell cycle, including master cyclins CDC28 and CLN1. Stress resistance transcription factor MSN4 and its upstream effector RIM15 are conspicuously up-regulated in the immobilized state, and an immobilized rim15 knockout strain fails to exhibit the long-lived, growth-arrested phenotype, suggesting that altered regulation of the Rim15-mediated nutrient-sensing pathway plays an important role in extending yeast chronological lifespan under calorie-unrestricted conditions. cell longevity | Ca-alginate encapsulation U nicellular microbes senesce and die (for reviews, see refs.

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“…The isolation and application of auxotrophic mutants is a cornerstone of yeast genetic studies (Mount et al, 1996;Pronk, 2002;Snow, 1966). Here, we used a novel approach to identify the nutrient requirements and genotypes of K. marxianus auxotrophic mutants (Kaiser et al, 1994 ).…”

Section: Auxotrophic Mutants In K Marxianusmentioning

confidence: 99%

Identification of auxotrophic mutants of the yeast Kluyveromyces marxianus by non‐homologous end joining‐mediated integrative transformation with genes from Saccharomyces cerevisiae

Yarimizu

Nonklang

Nakamura

et al. 2013

Yeast

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The isolation and application of auxotrophic mutants for gene manipulations, such as genetic transformation, mating selection and tetrad analysis, form the basis of yeast genetics. For the development of these genetic methods in the thermotolerant fermentative yeast Kluyveromyces marxianus, we isolated a series of auxotrophic mutants with defects in amino acid or nucleic acid metabolism. To identify the mutated genes, linear DNA fragments of nutrient biosynthetic pathway genes were amplified from Saccharomyces cerevisiae chromosomal DNA and used to directly transform the K. marxianus auxotrophic mutants by random integration into chromosomes through non-homologous end joining (NHEJ). The appearance of transformant colonies indicated that the specific S. cerevisiae gene complemented the K. marxianus mutant. Using this interspecific complementation approach with linear PCR-amplified DNA, we identified auxotrophic mutations of ADE2, ADE5,7, ADE6, HIS2, HIS3, HIS4, HIS5, HIS6, HIS7, LYS1, LYS2, LYS4, LYS9, LEU1, LEU2, MET2, MET6, MET17, TRP3, TRP4 and TRP5 without the labour-intensive requirement of plasmid construction. Mating, sporulation and tetrad analysis techniques for K. marxianus were also established. With the identified auxotrophic mutant strains and S. cerevisiae genes as selective markers, NHEJ-mediated integrative transformation with PCR-amplified DNA is an attractive system for facilitating genetic analyses in the yeast K. marxianus.

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Auxotrophic Yeast Strains in Fundamental and Applied Research

Cited by 267 publications

References 51 publications

Heterologous carotenoid production in Saccharomyces cerevisiae induces the pleiotropic drug resistance stress response

Heterologous carotenoid production in Saccharomyces cerevisiae induces the pleiotropic drug resistance stress response

Uncoupling reproduction from metabolism extends chronological lifespan in yeast

Identification of auxotrophic mutants of the yeast Kluyveromyces marxianus by non‐homologous end joining‐mediated integrative transformation with genes from Saccharomyces cerevisiae

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